Protein conformational isomers, methods of making, methods for using, compositions comprising and products made therewith

Abstract

Stable protein conformational isomers and methods for producing and isolating such isomers are disclosed. Methods of using such isomers, and products and compositions comprising such isomers are also disclosed.

1. A method for producing stable isomers of a protein, the method comprising:
 denaturing a sample comprising a recombinantly modified protein comprising at least one non-native cysteine in a denaturing buffer under incubation conditions sufficient to produce a mixture of stable isomer proteins, wherein said stable isomer proteins have a non- native conformation and comprise at least one non-native disulfide bond, isolating said stable isomer proteins, and screening the stable isomer proteins for use as a therapeutic agent, wherein the protein is not hirudin, tick anticoagulant peptide, potato carboxypeptidase inhibitor, or α-lactalbumin. 
 
     
     
       2. The method of  claim 1  wherein said buffer comprises a denaturant. 
     
     
       3. The method of  claim 2  wherein said denaturant is selected from the group consisting of urea, guanidinium chloride (GdmCl), guanidine thiocyanate (GdmSCN), organic solvents, elevated temperature, extreme pH, surfactants and detergents, and mechanical forces such as shaking, shearing, ultrasound, radiation and pressure. 
     
     
       4. The method of  claim 1  wherein said buffer comprises a thiol agent. 
     
     
       5. The method of  claim 4  wherein said thiol agent is selected from the group consisting of 2-mercaptoethanol, reduced glutathione, cysteine, and thiol-containing chemical compounds. 
     
     
       6. The method of  claim 1  wherein said incubation conditions comprise a temperature of between about 23° C. to about 37° C., and a time period ranging from about 15 minutes to about 7 days. 
     
     
       7. The method of  claim 1  wherein said recombinantly modified proteins comprises at least two non-native disulfide bonds. 
     
     
       8. The method of  claim 1  wherein said mixture comprises more than one species of stable isomer proteins, wherein each of the more than one species of stable isomer proteins has a species-specific non-native conformation, and wherein each of the more than one species of stable isomer proteins differs from one another by its species-specific non-native conformation. 
     
     
       9. A method for selectively making stable non-native protein isomers, the method comprising: denaturing a modified protein comprising at least one new disulfide bonding site in a denaturing buffer under incubation conditions sufficient to produce a mixture of stable modified protein isomers; isolating one or more of the stable modified protein isomers; and screening the isolated stable modified protein isomers for use as a therapeutic agent, wherein the protein is not hirudin, tick anticoagulant peptide, potato carboxypeptidase inhibitor, or α-lactalbumin. 
     
     
       10. The method of  claim 9 , wherein the buffer comprises a denaturant selected from the group consisting of urea, GdmCl, GdmSCN and organic solvents. 
     
     
       11. The method of  claim 9 , wherein the conditions comprise denaturing conditions selected from the group consisting of elevated temperature, extreme pH, surfactants and detergents, and mechanical forces such as shaking, shearing, ultrasound, radiation and pressure. 
     
     
       12. The method of  claim 9 , wherein the buffer comprises a thiol agent selected from the group consisting of 2-mercaptoethanol, reduced glutathione and cysteine. 
     
     
       13. The method of  claim 9 , wherein the incubation conditions comprise a temperature of between about 23° C. to about 37° C. 
     
     
       14. The method of  claim 9 , wherein the incubation conditions comprise a time period ranging from about 15 minutes to about 7 days. 
     
     
       15. The method of  claim 1 , wherein the mixture of stable isomer proteins comprises a desired stable isomer population, wherein the desired stable isomer population is produced by choosing specific incubation conditions. 
     
     
       16. The method of  claim 1 , wherein the mixture of stable isomer proteins comprises an agonist of the native protein. 
     
     
       17. The method of  claim 1 , wherein the mixture of stable isomer proteins comprises an antagonist of the native protein. 
     
     
       18. The method of  claim 1 , wherein specific incubation conditions are chosen, wherein the specific incubation conditions chosen produce a specific population of stable isomer proteins. 
     
     
       19. The method of  claim 9 , wherein specific incubation conditions are chosen, wherein the specific incubation conditions chosen produce a specific population of stable modified protein isomers. 
     
     
       20. The method of  claim 1 , wherein the stable isomer proteins are screened for use as a therapeutic agent for treatment of a conformational disease. 
     
     
       21. The method of  claim 1  further comprising treating a conformational disease with one or more of the stable isomer proteins identified as useful as a therapeutic agent. 
     
     
       22. The method of  claim 9 , wherein the stable modified protein isomers are screened for use as a therapeutic agent for treatment of a conformational disease. 
     
     
       23. The method of  claim 9  further comprising treating a conformational disease with one or more of the stable isomer proteins identified as useful as a therapeutic agent. 
     
     
       24. The method of  claim 5 , wherein said thiol agent is selected from, the group consisting of 2-mercaptoethanol, reduced glutathione, and cysteine. 
     
     
       25. A method for producing stable isomers of a protein, the method comprising:
 denaturing a sample comprising a starting protein in a denaturing buffer under incubation conditions sufficient to produce a mixture of stable isomer proteins, wherein the denaturing buffer comprises a thiol agent, wherein the incubation conditions leave the protein fully oxidized, wherein the stable isomer proteins have a non-native conformation, comprise at least one non-native disulfide bond that was not present in the starting protein, isolating the stable isomer proteins, and screening the stable isomer proteins for use as a therapeutic agent, 
 wherein the protein is not hirudin, tick anticoagulant peptide, potato carboxypeptidase inhibitor, or α-lactalbumin. 
 
     
     
       26. The method of  claim 25 , wherein the starting protein is a native protein having only native disulfide bonds. 
     
     
       27. The method of  claim 25 , wherein the protein comprises at least one non-native cysteine. 
     
     
       28. The method of  claim 25 , wherein the protein has at least one native cysteine replaced. 
     
     
       29. The method of  claim 25 , wherein a stable isomer protein is identified as a therapeutic agent. 
     
     
       30. The method of  claim 1 , wherein a stable isomer protein is identified as a therapeutic agent. 
     
     
       31. The method of  claim 9 , wherein a stable modified protein isomer is identified as a therapeutic agent. 
     
     
       32. A method for producing stable isomers of a protein, the method comprising:
 denaturing a sample comprising a recombinantly modified protein comprising at least one non-native cysteine in a denaturing buffer under incubation conditions sufficient to produce a mixture of stable isomer proteins, wherein said stable isomer proteins have a non- native conformation and comprise at least one non-native disulfide bond, wherein the incubation conditions leave the protein fully oxidized, isolating the stable isomer proteins, and screening the stable isomer proteins for use as a therapeutic agent, wherein the protein is not hirudin, tick anticoagulant peptide, potato carboxypeptidase inhibitor, or α-lactalbumin. 
 
     
     
       33. The method of  claim 1 , wherein the protein remains fully oxidized under the incubation conditions. 
     
     
       34. The method of  claim 9 , wherein the protein remains fully oxidized under the incubation conditions.